Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells

Abstract

Introduction: Mesenchymal stem cells (MSCs) are adult, multipotent, stem cells with immunomodulatory properties. The mechanisms involved in the capacity of MSCs to inhibit the proliferation of proinflammatory T lymphocytes, which appear responsible for causing autoimmune disease, have yet to be fully elucidated. One of the underlying mechanisms studied recently is the ability of MSCs to generate T regulatory (Treg) cells in vitro and in vivo from activated peripheral blood mononuclear cells (PBMC), T-CD4+ and also T-CD8(+) cells. In the present work we investigated the capacity of MSCs to generate Treg cells using T-CD4(+) cells induced to differentiate toward the proinflammatory Th1 and Th17 lineages.

Methods: MSCs were obtained from mouse bone marrow and characterized according to their surface antigen expression and their multilineage differentiation potential. CD4(+) T cells isolated from mouse spleens were induced to differentiate into Th1 or Th17 cells and co-cultured with MSCs added at day 0, 2 or 4 of the differentiation processes. After six days, CD25, Foxp3, IL-17 and IFN-γ expression was assessed by flow cytometry and helios and neuropilin 1 mRNA levels were assessed by RT-qPCR. For the functional assays, the 'conditioned' subpopulation generated in the presence of MSCs was cultured with concanavalin A-activated CD4(+) T cells labeled with carboxyfluorescein succinimidyl ester. Finally, we used the encephalomyelitis autoimmune diseases (EAE) mouse model, in which mice were injected with MSCs at day 18 and 30 after immunization. At day 50, the mice were euthanized and draining lymph nodes were extracted for Th1, Th17 and Treg detection by flow cytometry.

Results: MSCs were able to suppress the proliferation, activation and differentiation of CD4(+) T cells induced to differentiate into Th1 and Th17 cells. This substantial suppressive effect was associated with an increase of the percentage of functional induced CD4(+)CD25(+)Foxp3(+) regulatory T cells and IL-10 secretion. However, using mature Th1 or Th17 cells our results demonstrated that while MSCs suppress the proliferation and phenotype of mature Th1 and Th17 cells they did not generate Treg cells. Finally, we showed that the beneficial effect observed following MSC injection in an EAE mouse model was associated with the suppression of Th17 cells and an increase in the percentage of CD4(+)CD25(+)Foxp3(+) T lymphocytes when administrated at early stages of the disease.

Conclusions: This study demonstrated that MSCs contribute to the generation of an immunosuppressive environment via the inhibition of proinflammatory T cells and the induction of T cells with a regulatory phenotype. Together, these results might have important clinical implications for inflammatory and autoimmune diseases.

Figure 1

Surface antigens and differentiation potential of murine mesenchymal stem cells (MSCs). A) Phenotypic characterization of murine MSCs at passage nine to ten by FACS analysis. A representative histogram for each antigen is shown in red. B) Multilineage differentiation potential was assessed by their capacity to differentiate into chondrocyte (Col2 and aggrecan expression, up), adipocyte (Oil Red O staining; middle), and osteoblast (alizarin red S staining; bottom). FACS, fluorescence-activated cell sorting.

Figure 2

MSCs inhibit Th1 and Th17 differentiation depending on the stage of activation and cell ratio. The differentiation of Th1 and Th17 cells was measured by intracellular detection of IFN-γ and IL-17 positive cells, respectively, from purified CD4-T cells differentiated into Th1 and Th17 cells in the presence or absence of MSCs at different stages of activation and different MSC:Th ratios (left: representative dot plot: 1:10 upper panels and 1:100 lower panels). ** = P <0.01 and * = P <0.05, n = six for Th1 and n = seven for Th17. Values represent means ± SED for six and seven independent experiments for Th1 and Th17 cells, respectively. MSCD0, D2 and D4 = MSC added at day 0, 2 and 4 of the differentiation process, respectively. MSCs, mesenchymal stem cells; Th, T helper.

Figure 3

MSCs inhibit the activation and the proliferation of Th1 and Th17 cells.A) MSCs inhibit the expression of CD25 in the early stage of activation of Th1 and Th17 cells. T cell activation was measured using purified CD4⁺ T cells differentiated into Th1 or Th17 cells in the presence or absence of MSCs according to the expression of the activation surface antigen CD25 (left: representative dot plot: 1:10 upper panels and 1:100 lower panels). B) MSCs suppress Th1 and Th17 cells independent of the stage of activation and cell ratio. Relative cell quantification was determined using purified CD4⁺ T cells differentiated into Th1 or Th17 cells in the presence or absence of MSCs added at day 0, 2 or 4 after T cell activation and at a MSCs:Th ratio (1:10 and 1:100), using the Cell Titer Glo™ luminescence assay. RLU = relative light units. MSCs, mesenchymal stem cells; Th, T helper.

Figure 4

MSCs generated iTreg cells during the differentiation process of Th1 and Th17 cells. A) Treg generation from Th1 and Th17 cells cocultured with MSCs at different stages of activation and MSC:Th ratios. Treg phenotype was determined according to the percentage of CD4 + CD25+ cells that express Foxp3 by flow cytometry (Dot plot upper A for Th1 and Upper B for Th17). B) Foxp3, Nrp 1 and helios mRNA expression levels weres quantified from Th1 and Th17 cells cocultured in the presence or absence of MSCs at different stage of activation and MSC:Th ratios (** = P <0.01 and * = P <0.05 ). iTreg, induced Treg; MSCs, mesenchymal stem cells; Nrp 1, neuropilin 1; Th, T helper; Treg, regulatory T cells.

Figure 5

IL-10 production from supernatants of coculture of Th1 and Th17 cells with MSCs and functional assay.(A) IL-10 production was quantified in the supernatants of Th1 and Th17 cells co-cultured in the presence or absence of MSCs. * = P <0.05, comparing to Th1 and Th17 cells as control group. All the values represent means ± SED of three independent experiments. B) Functional effect of iTreg cells generated using Th1 and Th17 cells in cocultures with MSCs (conditioned Th1 or Th17, Th1^cond or Th17^cond, respectively). The suppressive capacity of Th1^cond or Th17^cond was assessed by following the number of cell divisions of ConA-stimulated T cells, which were labeled by CFSE. Different ratios of Th^cond:CD4⁺ (1:1; 1:2 or 1:5) were tested. ** = P <0.01 and * = P <0.05. All the values represent means ± SED of three independent experiments for Th1 and Th17 cells. Th1^cond and Th17 ^cond = Th1 and Th17 cells that were co-cultured with MSCs added at different time points of the differentiation processes. CFSE, carboxyfluorescein succinimidyl ester; ConA, concanavalin A; iTreg, induced regulatory T cells; MSCs, mesenchymal stem cells; Th, T helper.

Figure 6

PGE2, IL-10 and TGF-β1 expression on MSCs co-cultured with Th1 and Th17 cells. A) PGE2 was quantified in the supernatants of MSCs co-cultured in the presence or absence of Th1 and Th17 cells. B) IL-10 and TGF-β1 expression was quantified in the supernatants of MSCs cultured in contact with Th1 and Th17 cells at different stages of the differentiation process (Day 0 and 4) ** = P <0.01, * = P <0.05, comparing to MSCs as the control group. All the values represent means ± SED of three independent experiments. MSCs, mesenchymal stem cells; PGE2, prostaglandin E2; TGF-β1, transforming growth factor β; Th, T helper.

Figure 7

Therapeutic effects and Th1, Th17 and CD4 + CD25 + Foxp3 + Treg populations in experimental autoimmune encephalomyelitis mice treated with MSCs. A) Early intravenous injection of MSCs ameliorates EAE. MSCs were injected at days 18 and 30 after immunization as shown by arrows (1 × 10⁶ MSC/mice). B) CD4⁺IL17⁺ and CD4⁺IFN-γ⁺ cells in lymph nodes. MSCs significantly reduced the percentage of Th17 cells when injected at day 18 of the disease. C) CD4⁺CD25⁺Foxp3⁺ T cells in lymph nodes. MSCs induce CD4⁺CD25⁺Foxp3⁺ cells when injected at day 18 of the disease. Control: EAE mice without treatment. MSCD18: EAE mice treated with MSCs injected 18 days post-immunization. MSCD30: EAE mice treated with MSCs injected 30 days post-immunization. * = P <0.05, compared to EAE untreated mice as control group. All the values represent means ± SED of n = 5 control condition and n = 6 for MSCs treated mice. EAE, experimental autoimmune encephalomyelitis; MSCs, mesenchymal stem cells; Th, T helper; Treg, regulatory T cells.